Preparation of Anti—Cardiac Troponin I Monoclonal Antibodies and Their Characterization with Surface Plasmon Resonance Biosensor

IntroductionIn fast and slow skeletal and cardiac muscles,troponin I,the inhibitory protein of the troponin-tropomyosin complex,exists in three isotype formsencoded by three separated genes.The amino acidsequences of the two skeletal and one cardiac Tn Iforms( s Tn I and c Tn I,respectively) exhibit40 %dissimilarity[1] .Moreover,human cardiac Tn I has31 additional residues on the N - terminal end,which do not exist in skeletal forms,thus it pro-vides a high potential for obtaining cardiac-…     

Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for monensin.

Monensin, a member of the ionophoric polyether antibiotics, is used primarily as a coccidiostat. A protein conjugate of monensin was prepared and utilized to produce monoclonal antibodies in the BALB/c-P3X63Ag8U.1 fusion system. Only one hybridoma that produces monoclonal antibody against monensin was isolated from one in 329 wells. The monoclonal antibody was used to develop quantitative assays for monensin by means of an enzyme-linked immunosorbent assay (ELISA). The detection limit was 1 ng ml-1 and the relative standard deviations were 2.1-6.3% intra-assay and 5.9-12.9% inter-assay. All ELISA results for assay of chicken plasma and cattle milk were confirmed using a bioassay to be used as the official method. The ELISA and bioassay results showed close correlations for plasma (r2 = 0.98, n = 25) and milk (r2 = 0.95, n = 25). Using the anti-monensin monoclonal antibodies produced, a rapid test kit based on the immunochromatographic method was developed. Detection limits of monensin for cattle milk, cattle plasma and chicken plasma were about 40, 40 and 160 ppb. respectively.     

化学发光免疫分析法检测血清中前列腺特异抗原复合物

采用杂交瘤技术构建抗前列腺特异抗原-α1-抗胰凝乳蛋白酶( Prostate specific antigen-α1-antichymot-rypsin complex,PSA-ACT)单克隆抗体(单抗)细胞株,共获得8株稳定分泌PSA-ACT单抗的细胞株,纯化后进行免疫特性鉴定并做单抗配对。选择其中一对单抗,采用辣根过氧化物酶( HRP)催化鲁米诺( lumino)-H2 O2化学发光体系,建立了测定人血清中PSA-ACT浓度的化学发光酶免疫分析方法( CLEIA)。对包被缓冲液、包被抗体浓度、酶标抗体稀释度、温育时间、发光反应时间和免疫反应步骤等实验参数进行了优化。在最佳实验条件下, PSA-ACT浓度在5~40 ng/mL范围内线性关系良好(R2=0.9943),检出限为0.53 ng/mL,批内相对标准偏差(RSD)为4.6%~6.6%,批间RSD为5.7%~8.0%,回收率为95.4%~104.2%,与游离前列腺特异性抗原F-PSA的交叉反应率为0.6%。本方法简单、稳定、灵敏、快速,为开发检测PSA-ACT的CLEIA试剂盒奠定了基础。     

Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate: application to enzyme-linked immunosorbent assay for urinary and serum cortisol.

Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype gamma1, kappa) showed an affinity constant (K(a)) for cortisol of 1 x 10(9) M(-1) and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17alpha-hydroxyprogesterone (12%), 6beta-hydroxycortisol (8.4%), and tetrahydrocortisol (< 0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 +/- 17.0 microg/day (n = 7) and 80.8 +/- 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.     

Effect of α-Ketoglutarate on Monoclonal Antibody Production of Hybridoma Cell Lines in Serum-Free and Serum-Containing Medium

Process development and optimization for increase population growth and protein productivity in mammalian cell culture have been studied for many years. In this study, the behavior of hybridoma cells was investigated using six-well micro-titer plate systems with a working volume of 4 ml. Mouse hybridoma cell lines D2 and 2C83G2 were seeded in serum-free and serum-containing media and cultured for 8 days. alpha-Ketoglutarate is an integral component of the tricarboxylic acid (TCA) cycle and is produced from glutamine via glutamate. To study its effect on cell growth, metabolism, and monoclonal antibody (mAb) production, 2 mM alpha-ketoglutarate (pH 7.2) was added in both media at the beginning of the cultivation and in another set after 72 h. High cell density was observed in D2 cell culturing in serum-free medium, while 2C83G2 cell line showed high cell density in serum-containing medium. However, both cell lines cultured in serum-free medium gave viability above 70% when grown for 8 days. The supplement of 2 mM alpha-ketoglutarate supported cell growth and mAb production of both hybridoma cell lines in serum-free and serum-containing medium. The addition of alpha-ketoglutarate at the beginning of the batch cultivation gave better result in cell growth and mAb production as compared to alpha-ketoglutarate supplementation after 72 h. However, addition after 72 h was better than no addition at all. This indicates that alpha-ketoglutarate have a positive effect on production and release of antibody.     

Trace analysis of microcystins in water using enzyme-linked immunosorbent assay

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin (KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. In the ELISA test, a microtiter plate coated with MCLR-bovine serum albumin conjugate was incubated with standard microcystin samples. The amount of antibody bound was determined by the reaction of peroxidase-labeled anti-mouse IgG with its substrate, 3,3′,5,5′-tetramethyl benzidine (TMB). Since the ELISA test was highly sensitive, the newly developed ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 30 and 1600 pg/mL.     

Assay of cell surface-bound immunoliposomes using monoclonal antibody reactive with a cross-linking reagent.

A monoclonal antibody (mAb) reactive with a crosslinking reagent, N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine (mMBPE), in liposomes was produced from a hybridoma clone established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells from a BALB/c mouse hyperimmunized with the antibody-coated liposomes containing mMBPE. Using this mAb (termed AL-6), the quantity of immunoliposomes bound on target tumor cells was assessed by flow cytofluorometry. The results obtained using fluorescein isothiocyanate-coupled AL-6 allowed the enumeration not only of the immunoliposomes bound on all tumor cells but also those on individual target tumor cells. The relevance of this assay method was confirmed by a comparison with another assay method of cell-bound liposomes using immunoliposomes containing carboxyfluorescein in the vesicles.     

Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5–7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2 l hybridoma supernatants.     

Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease

Hybridoma clones secreting specific monoclonal antibodies (mAb) against the bacterium-like organism associated with citrus greening disease were produced from homogenates of infected pholem tissues used as immunogens in mice immunizations.Differential ELISA and immunofluorescence were used to screen for hybridomas secreting mAb against the greening organism (GO). Two such hybridoma clones were obtained. The mAb were specific for the GO. No cross-reactions were seen with any of the other phloem-limited prokaryotes tested nor with healthy plant material.     

Recovery of C-Phycocyanin in the Presence of Cells Using Expanded Bed IEC

In order to study the relationship between spinal cord injury and the change of nerve growth factor (NGF), an analytical method for NGF by capillary electrophoresis-based immunoassay (CEIA) with laser-induced fluorescence (LIF) was developed. Having been dissolved in phosphate buffer solution and concentrated with vacuum freeze-drying, NGF in spinal cord of rat was allowed to react with NGF monoclonal antibody labeled with fluorescence isothiocyanate (FITC). Then the immuno-complex, FITC-labeled anti-NGF and FITC were separated and determined by LIF-CEIA using Kiton red as the internal standard. The linear range of the method was 2?C30?ng?mL^?1 and the limit of detection was 0.35?ng?mL^?1. The relative standard derivations for relative migration time and relative fluorescence intensity ratio were 7.98% and 6.52%, respectively. The contents of NGF from spinal cord of rat were determined by both the proposed method and Western blotting. The results with the two methods agreed well. The spiked recoveries of the samples were 88.5?C116.3%. The proposed method was rapid, precise and inexpensive.     

量子点标记的斑点免疫渗滤分析定量检测cTnI

利用量子点良好的光谱特征和光化学稳定性, 结合免疫分析技术, 对心肌肌钙蛋白I(cTnI)特异性进行定量检测. 用量子点标记cTnI的单克隆抗体(2F11), 通过SDS-PAGE电泳证明标记成功. 斑点免疫膜渗滤法证明标记后的2F11仍具有良好的生物学活性, 再将标记并纯化后的2F11与NC膜上不同浓度的cTnI进行免疫反应, 使用ImageMaster图像分析软件对膜上荧光斑点图像进行定量分析. 应用此方法测得cTnI的浓度和斑点处相对荧光值有良好的线性关系(R2=0.9966), 最低检出值为120 ng.     

Monoclonal antibody-mediated clean-up procedure for the high-performance liquid chromatographic analysis of chloramphenicol in milk and eggs

A simple, rapid and specific sample preparation method based on antibody-mediated clean-up for the determination of chloramphenicol (CAP) in milk and eggs was developed. Skimmed milk and centrifuged egg homogenates were filtered and directly applied to immunoaffinity columns which were prepared by coupling monoclonal antibodies against CAP to a carbonyldiimidazole-activated support. Using a 0.2 M glycine, 0.5 M NaCl (pH 2.8) solution as an eluent, the immunoaffinity columns can be used more than 30 times without a decrease in column capacity. In subsequent high-performance liquid chromatographic analysis, no matrix interferences were observed. Good recoveries were obtained at spiking levels of 1-100 micrograms kg-1. Due to the high specificity of the clean-up procedure, the limit of detection can be lowered by increasing the test portion. Concerning milk, the limit of detection was successfully lowered to 20 ng kg-1 by increasing the test portion to 11 (recovery 99%). The method was applied to eggs produced by hens treated with CAP. The results are compared with those obtained by solid-phase extraction using silica gel.     

Characterization of a New Monoclonal Antibody Against Mercury(II)

Monoclonal antibodies (mabs) were produced against mercury (II) and an enzyme immunoassay was developed for the detection of mercury (II) in water. Since mercury (II) ions are too small to elicit an immune response, they were coupled via glutathione (GSH) to the immunogenic carrier protein keyhole limpet hemocyanin (KLH). Several mice were immunised with this KLH-GSH-Hg immunoconjugate. Spleen cells of immunised mice were fused with myeloma cells. The resulting hybridoma cells were screened for the production of specific anti-Hg mabs. Five positive cells were detected. The hybridoma cell line K3C6 was adjusted to protein free medium; it produced mabs with high selectivity and sensitivity. A detection limit of 2.8 μg/L HgCl2 (= 2.1 μg/L Hg²⁺) was achieved with a non-competitive enzyme immunoassay (EIA). Cross-reactivities with other metals were below 1%. Measurement of spiked water samples with this EIA showed good correlation with results obtained by mass spectrometry with inductive coupled plasma (ICP-MS).     

氟甲喹单克隆抗体制备、鉴定及间接竞争酶联免疫吸附分析法

以氟甲喹(FLU)为原料,合成4个碳原子手臂的半抗原(FLUABA),采用活泼酯法与牛血清白蛋白(BSA)偶联制备免疫抗原,通过免疫Balb/c小鼠及细胞融合,获得1株稳定分泌抗氟甲喹单克隆抗体的杂交瘤细胞株DB6-E7,其抗体亚类为IgG1,亲和力常数(KA)为8.19×108L/mol。将氟甲喹、FLUABA及6个碳原子手臂的半抗原FLUACA分别与卵清白蛋白(OVA)偶联作为包被抗原,研究异源包被对间接竞争ELISA灵敏度的影响。结果表明,异源包被可显著提高ELISA方法的灵敏度。基于最佳异源包被(FLU-OVA)的酶联免疫吸附分析法的IC50为26.33μg/L,检出限为4μg/L,定量检测范围为8.0～114μg/L(IC20～IC80)。与喹诺酮类药物及结构类似物几乎不存在交叉反应,特异性高。此方法可满足畜禽产品中氟甲喹残留的快速筛查。     

Monoclonal antibody to 2-amino-3-methylimidazo(4,5-F)quinoline,a dietary carcinogen

In order to investigate relationships between human carcinogenesis and dietary carcinogens, one hybridoma cell line secreting a monoclonal antibody against 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), a dietary carcinogen, was produced by fusing splenocytes from Balb/c mice immunized with IQ-Lysine(Lys)-Ascaris protein conjugate. The subclass of monoclonal anti-IQ antibody was determined by double immunodiffusion using culture medium and identified as IgG1. Monoclonal anti-IQ antibody was purified from ascites fluids of Balb/c mice with affinity chromatography on Protein A-Sepharose CL4B and analyzed concerning its cross-reactivity and sensitivity with RIA. Finally, we showed that our monoclonal antibody recognized IQ, 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ) and several beta-carbolines more intensely and that the sensitivity to IQ was 23 nmol in 50% displacement.     

Rapid identification of immunostimulatory alpha-galactosylceramides using synthetic combinatorial libraries

Two 60+-membered libraries of alpha-galactosylceramides have been prepared by reactions between activated ester resins and two core, fully deprotected galactosylated sphingoid bases. The libraries were evaluated for their ability to stimulate CD1d-restricted NKT cells, using in vitro stimulation of a murine NKT cell hybridoma line and for their ability to induce the expansion of NKT cells from peripheral blood mononuclear cells (PBMC) of a normal human subject. Our results showed that many compounds constructed on a C18-phytosphingosine base had significant stimulatory activity in both assays. Because no product purification was required, this approach is particularly attractive as a method for rapid synthesis of large libraries of potential immunomodulatory glycosylceramides.     

Development of solid-phase microextraction followed by gas chromatography-mass spectrometry for rapid analysis of volatile organic chemicals in mainstream cigarette smoke

In this work, a novel, simple and efficient method based on solid-phase microextraction (SPME) followed by gas chromatography-mass spectrometry (GC-MS) was developed to the analysis of volatile organic chemicals (VOCs) in mainstream cigarette smoke (MCS). Using a simple home-made smoking machine device, extraction and concentration of VOCs in MCS were performed by SPME fiber, and the VOCs adsorbed on fiber were desorbed, and analyzed by GC-MS. The extraction fiber types and the desorption conditions were studied, and the method precision was also investigated. After the investigation, the optimal fiber was divinylbenzene/carboxen/polydemethylsiloxane (DVB/CAR/PDMS), and the optimal desorption condition was 250 degrees C for 3 min. The method precision was from 2% to 11%. Finally, the proposed method was tested by its application of the analysis of VOCs in MCS from 10 brands of cigarettes and one reference cigarette. A total of 70 volatile compounds were identified by the proposed method. The experimental results showed that the proposed method was a simple, rapid, reliable, and solvent-free technique for the determination of VOCs in MCS.     

Immobilized enzymes and cells in poly(N-vinyl caprolactam)-based hydrogels

A one-step mild method for entrapping animal cells and enzymes in macroporous composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogels is described. Some properties of immobilized enzymes, such as thermal and storage stabilities and stability in water/organic media were investigated. Composite PVCL-CaAlg gels were successfully applied to immobilize a number of proteases, namely, trypsin, α-chymotrypsin, carboxypeptidase B, and thrombin. Thermal stability of the immobilized preparations obtained by entrapment in hydrogel beads allowed us to use them at 65–80†C, while the native enzymes were completely inactivated at 50–55°C. Various applications of enzymes and cells immobilized in beads weredemonstrated. Immobilized trypsin and carboxypeptidase B were applied to prepare human insulin from recombinant proinsulin. The hydrogel beads with entrapped α-chymotrypsin were used in enantioselective hydrolysis of Shiff's base of D,L-phenylalanine ethyl ester (SBPH) in acetonitrile/water medium. Thrombin immobilized in PVCL-based hydrogel films was shown to be a promising compound for wound treatment. To prepare pure preparations of monoclonal antibodies (MAb) several hybridoma cell lines, including hybridoma cell lines producing MAb to interleukin-2, were successfully cultivated in the hydrogel beads.